The abbreviations to be used in the application documents are as follows.    BSA: bovine serum albumin    EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide    EDNA: ethylenediamine tetraacetic acid    ELISA: enzyme-linked immunosorbent assay    GlcA: glucuronic acid    GlcN: glucosamine    GlcNAc: N-acetylglucosamine    HEPES: 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid    HP: heparin    HPLC: high-performance liquid chromatography    HRP: horseradish peroxidase    HS: heparan sulfate    IgG: immunoglobulin G    MES: 2-morpholinoethanesulfonic acid    NAH: N-acetyl heparosan    ND: N-deacetylase    NDST: N-deacetylase/N-sulfotransferase    PAPS: 5′-phosphoadenosine 3′-phosphosulfate    PBS: phosphate buffered saline    PCR: polymerase chain reaction    PDP: 2-pyridyldisulfidepropionyl    RIA: radioimmunoassay    SDS: sodium dodecyl sulfate    SPDP: N-succinimidyl-3-(2-pyridylthio)propionate    SS bond: disulfide bond    ST: N-sulfotransferase    TBS: Tris-HCl buffered saline    TMB: 3,3′,5,5′-tetramethylbenzidine
NDST is an enzyme which is concerned in the synthesis of HP and HS, and is an enzyme that has both of the ND activity and ST activity. It has been reported that 4 species of variants (NDST 1, NDST 2, NDST 3 and NDST 4) are present in NDST and their substrate specificity and the like are different from one another. For example, ND activity of NDST 3 is high and ST activity thereof is low. On the other hand, ND activity of NDST 4 is low and ST activity thereof is markedly high (Non-patent Reference 1).
It has been reported that NDST 1 knockout mice are newborn-lethal. On the other hand, although NDST 2 knockout mice do not show lethality, reduction of granules inside the mast cell, reduction of HP negative charge, reduction of the histamine content to 1/170 and the like changes have been reported (Non-patent References 2 to 8). Thus, there is a possibility that NDST is concerned in the diseases caused by such phenomena in the living body.
Regarding the method for measuring NDST activity, a measuring method which uses a substrate labeled with a radioisotope is known. According to the method, using NAH in which the acetyl group of GlcNAc residue is labeled with a radioisotope (3H) as a substrate, the ND activity of NDST is measured by measuring the amount of CH3COO3H released from the substrate by the enzymatic activity. Additionally, the ST activity of NDST is measured using PAPS labeled with a radioisotope (35S) and N-desulfated HP or N-desulfated HS, by measuring the incorporated amount of N-sulfate (35S) of said polysaccharide by the enzymatic activity (Non-patent References 1, 12 to 15, Patent Reference 1).
Additionally, although the methods for measuring NDST activity which do not use radioisotope are also known, only a sandwich ELISA method which uses a monoclonal antibody JM403 (Non-patent References 9 and 10) and an HPLC method which uses heparinase digests of the reaction product have been reported.
Although the former measuring method is a method which uses the reaction of de-N-acetylated region formed by ND with the antibody JM403, it requires two or more epitopes since it is a sandwich method. Additionally, the ST activity cannot be detected by the method (Non-patent Reference 11).
Although the latter measuring method is a method in which de-N-acetylation degree and N-sulfation degree of the de-N-acetylated NAH formed by the ND activity and ST activity and the unsaturated disaccharide obtained by the heparinase digestion of N-sulfated NAH are calculated using the “2-8 structural analysis as a combination of glycosaminoglycan degrading enzyme and HPLC” described in Non-patent Reference 16, it is not suited for chromatography, screening and the like methods for measuring activity of multiple samples (Patent Reference 2, Non-patent Reference 17).
When activities of ND, ST and NDST can be measured conveniently and quickly with high sensitivity and high accuracy and also at a moderate price, not only the detection, morbid state understanding and the like of diseases in which such enzymes are concerned and risks thereof can be easily carried out, but also screening and the like of substances which provide changes in the activities of said enzymes (inhibitors, activators and the like) can be carried out efficiently.    Patent Reference 1: US 2003/0109501A    Patent Reference 2: US 2004/0043447A    Non-patent Reference 1: Aikawa, J. et al., 2001, Journal of Biological Chemistry, vol. 276, no. 8, p. 5876-5882    Non-patent Reference Z: Humphries, D. E. et al., 1999, Nature, vol. 400, p. 769-772    Non-patent Reference 3: Forsberg, E. et al., 1999, Nature, vol. 400, p. 773-776    Non-patent Reference 4: Ringivall, M. et al., 2000, Journal of Biological Chemistry, vol. 275, no. 34, p. 25926-25930    Non-patent Reference 5: Pikas, D. E. et al., 2000, Biochemistry, vol. 39, p. 4552-4558    Non-patent Reference 6: Fukuda, M. et al., 2001, Journal of Biological Chemistry, vol. 276, no. 51, p. 47747-47750    Non-patent Reference 7: Esko, J. D. et al., 2001, Journal of Clinical Investigation, vol. 108, p. 169-173    Non-patent Reference S: Forsberg, E. et al., 2001, Journal of Clinical Investigation, vol, 108, p. 175-180    Non-patent Reference 9: van den Born, J. et al., 1992, Kidney International, vol. 41, p. 115-123    Non-patent Reference 10: van den Born, Y. et al., 1995, Journal of Biological Chemistry, vol. 270, no. 52, p. 31303-31309    Non-patent Reference 11: van den Born, J. et al., 2003, Glycobiology, vol. 13, no. 1, p. 1-10    Non-patent Reference 12: Brandan, E. et al, 1988, Journal of Biological Chemistry, vol. 263, no. 5, p. 2417-2422    Non-patent Reference 13: Bame, K. J. et al., 1991, Journal of Biological Chemistry, vol. 266, no. 16, p. 10287-10293    Non-patent Reference 14: Bame, K. J. et al., 1991, Journal of Biological Chemistry, vol. 266, no. 19, p. 12461-12468    Non-patent Reference 15: Verdugo, D. E. et al., 2002, Analytical Biochemistry, vol. 307, p. 330-336    Non-patent Reference 16: Shin Seikagaku Jikken Koza 3, Toshitsu II (published by Tokyo Kagaku Dojin, 1991), p. 49-62    Non-patent Reference 17: Saribas, A. S. et al., 2004, Glycobiology, vol. 14, no. 12, p. 1217-1228